The long range goal of this project is to use a novel adeno-associated virus (AAV) vector encoding therapeutic genes as a treatment against HIV. HIV infection remains a major blight on human society worldwide. Because attempts to control the spread of HIV and its persistence upon infection have yielded few successes, innovative approaches are urgently needed. Gene-based therapeutics offer hope to both prevent and cure HIV infection, potentially simultaneously, depending on the genetic elements incorporated. Recent clinical trials have shown that AAV is extremely safe and efficacious when the disease target is matched with AAV-serotype tissue tropism. To date, however, no AAV with sufficient efficiency has been described for therapeutic alteration of the primary HIV target, T cells. Directed evolutionary approaches using molecular techniques have the potential to create designer AAV vectors. Based on the work of ourselves and others, we hypothesize that directed evolution can be used to create novel AAV capsids capable of efficiently infecting T cells, and in particular, T cells already infected by HIV. Furthermore, we hypothesize that the evolved AAVs can then be engineered to deliver a therapeutic gene against HIV. This hypothesis will be tested as follows: Aim 1. To determine whether directed evolution of the AAV capsid gene can generate viruses with enhanced gene transfer efficiency in uninfected and HIV-infected lymphocytes. We will combine PCR- based mutagenesis with genetic shuffling to obtain hybrid AAV cap genes, and the resulting library of novel viruses will be selected for the ability to transduce H9 T cells or chronically HIV-infected H9 T cells. Selection on HIV-H9 cells may yield AAV capsids that can specifically infect HIV-infected cells, or both infected and uninfected cells. To isolate capsids that are specific for HIV-infected cells, negative selection will be performed using parental uninfected H9 T cells. Novel capsid variants will then be investigated for transduction efficiency in parental- and HIV-H9 T cells. Aim 2. To investigate whether novel AAV viruses can mediate gene transfer in order to protect naive lymphocytes and/or alter productive HIV infection of acutely or chronically infected lymphocytes. Successful AAVs will be evaluated for HIV-directed shRNA gene transfer approaches, with and without small molecule HIV inhibitors. The discovery of novel AAV capsids able to selectively infect HIV-infected cells offers the potential not only for new gene therapy vectors, but also for the selection and future identification of new cell surface epitopes or biomarkers useful for HIV detection and/or therapeutic intervention. The proposed research also provides an opportunity to investigate the complex consequences of viral co-infection and may lead to novel insights into viral interactions, pathogenicity, and therapeutic strategies.